CAJ | 학술논문

目的研究肠道病毒71型(EV71)感染人横纹肌肉瘤(RD)细胞后不同时期凋亡相关基因差异表达及信号通路转导机制.方法锥虫蓝染色观察EV71感染RD细胞的存活率,Annexin V-FITC/PI双染色法检测病毒感染后凋亡细胞形态学及凋亡发生率,PCR芯片分析病毒感染RD细胞8h和20h后88个凋亡相关基因的表达情况.结果 EV71(MOI=5)感染RD细胞8h后存活率显著下降.流式细胞仪检测显示病毒感染20 h后早期凋亡及晚期凋亡细胞分别上升至18.0％和19.1％.PCR芯片检测88个凋亡相关基因,EV71感染8h后,除IFN-β1上调5.22倍外,ACIN1、Akt、Apaf1、caspase、CIDEB等47个基因发生明显下调.病毒感染20 h后,FasL、CD40L、TNF、caspase-10、caspase-3等28个基因发生2倍以上上调.结论 EV71感染早期RD细胞凋亡相关基因表达下调与病毒凋亡抑制效应相关,而感染晚期病毒可激活Fas/FasL、TNF/TNFR死亡受体信号通路诱导细胞凋亡.同时宿主细胞也通过调节CD40/CD40L、NF-κB/RelA、PI3 K/Akt等信号通路途径抑制凋亡作用.
목적 연구장도병독71형(EV71)감염인횡문기육류(RD)세포후불동시기조망상관기인차이표체급신호통로전도궤제.방법 추충람염색관찰EV71감염RD세포적존활솔,Annexin V-FITC/PI쌍염색법검측병독감염후조망세포형태학급조망발생솔,PCR심편분석병독감염RD세포8h화20h후88개조망상관기인적표체정황.결과 EV71(MOI=5)감염RD세포8h후존활솔현저하강.류식세포의검측현시병독감염20 h후조기조망급만기조망세포분별상승지18.0％화19.1％.PCR심편검측88개조망상관기인,EV71감염8h후,제IFN-β1상조5.22배외,ACIN1、Akt、Apaf1、caspase、CIDEB등47개기인발생명현하조.병독감염20 h후,FasL、CD40L、TNF、caspase-10、caspase-3등28개기인발생2배이상상조.결론 EV71감염조기RD세포조망상관기인표체하조여병독조망억제효응상관,이감염만기병독가격활Fas/FasL、TNF/TNFR사망수체신호통로유도세포조망.동시숙주세포야통과조절CD40/CD40L、NF-κB/RelA、PI3 K/Akt등신호통로도경억제조망작용.
Objective To explore the different apoptotic gene expressions and apoptotic signaling transduction of human rhabdomyosarcoma (RD) cells infected by enterovirus 71 (EV71) in different stage.Methods The survival of EV71-infected RD cells was observed by trypan blue staining.The apoptotic morphology and rates of RD cells were surveyed and detected by Annexin V-FITC/PI staining and flow cytometry,respectively.PCR array was employed to analyze 88 apoptotic gene expressions from EV71-infected RD cells at 8 h and 20 h postinfection (p.i),respectively.Results After RD cells were infected with EV71 (MOI =5) at 8 h p.i,the viability was significantly decreased.Flow cytometry data demonstrated that the apoptotic rates of EV71-infected RD cells had increased to 18.0％ and 19.1％ at 20 h p.i in early and later stage respectively.RT-PCR array studies revealed significant variations in the expression of apoptotic genes.Among 88 genes,only the expression of IFN-β1 was upregulated 5.22 folds,whereas 47 genes including ACIN1,Akt,Apaf1,caspase and CIDEB were found to be downregulated that were lower than 2 folds at 8 h p.i.However,28 genes including FasL,CD40L,TNF,caspase-10 and caspase-3 were induced more than 2 folds after EV71 infection at 20 h.Conclusion The downregulation of apoptosis-related genes is associated with viral apoptosis-suppressing effect in RD cells in the early stage of EV71 infection.The death receptor signaling pathways including Fas/FasL and TNF/TNFR are activated to induce cell apoptosis in the late stage of EV71 infection.Moreover,host cell can also inhibit apoptosis by regulating signal pathway of CD40/CD40L,NF-κB/RelA and PI3K/Akt activation.